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12.4: Self‑splicing by group I introns (pre‑rRNA of Tetrahymena) - Biology

12.4: Self‑splicing by group I introns (pre‑rRNA of Tetrahymena) - Biology


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An in vitro reaction was established to examine the removal of an intron from the precursor to rRNA in Tetrahymena. Suprisingly, it was discovered that the splicing of the pre-RNA occurred in the absence of any added protein!

Further investigation revealed that the reaction requires a guanine nucleotide or nucleoside with a 3'‑OH, plus mono‑ and divalent cations. GTP, GDP, GMP or guanosine will work to initiate splicing. There is no requirement for protein or high energy bond cleavage

Self‑splicing occurs by a phosphoester transfer mechanism (Figure 3.3.11)

The 3'‑OH of the guanine nucleotide is the nucleophile that attacks and joins to the 5' phosphate of the first nucleotide of the intron. This leaves the 3'‑OH of the last nucleotide of the upstream exon available to attack and join the 5' phosphate of the first nucleotide of the downstream exon. These two phosphoester transfers result in a joining of the two exons and excision of the intron (with the initiating G nucleotide attached to the 5' end.) The excised intron is then circularized by attack of the 3'‑OH of the last nucleotide of the intron on the phosphate between the 15th and 16th nucleotides of the introns. Further degradation effectively removes the intron from the reaction and helps prevent the reverse reaction from occurring. Note that the phosphoester transfers are readily reversible unless the products (excised intron) are removed. There is no increase or decrease in the number of phosphoester bonds during this splicing.

The Intron is the Catalyst for Splicing in this System

RNA involvement in self‑splicing is stoichiometric, but the excised intron does have a catalytic activity in vitro. After a series of intramolecular cyclization and cleavage reactions, the linear excised intron lacking 19 nucleotides (called L-19 IVS) can be used catalytically to add and remove nucleotides to an artificial substrate. For instance, C5, which is complementary to the internal guide sequences of the intron, can be converted to C4 + C6 and other products (Figure 3.3.12).

The 3‑D structure of the folded RNA is responsible for the specificity and efficiency of the reaction (analogous to the general ideas about proteins with enzymatic activity). The specificity of splicing is caused, at least in part, by base‑pairing between the 3' end of the upstream exon and a region in the intron called the internal guide sequence. The initiating G nt also binds to a specific site in the RNA close to the 5' splice site. Thus two sites in the pre-rRNA intron are used sequentially in splicing (Figure 3.3.13 A and 3.3.13.B.).

Figure 3.3.13.A.

The internal guide sequence (IGS) is not not required for catalysis but does confer specificity. Thus one can design RNAs for exon exchange in cells. This potential avenue for therapy for genetic disorders is called "exon replacement therapy.


The ribosomal DNA region of the myxomycete Fuligo septica was investigated and found to contain 12 group I introns (four in the small subunit and eight in the large subunit ribosomal RNAs). We have performed molecular and phylogenetic analyses to provide insight into intron structure and function, intron-host biology, and intron origin and evolution. The introns vary in size from 398 to 943 nt, all lacking detectable open reading frames. Secondary structure models revealed considerable structural diversity, but all, except one (subclass IB), represent the common group IC1 intron subclass. In vitro splicing analysis revealed that 10 of the 12 introns were able to self-splice as naked RNA, but all 12 introns were able to splice out from the precursor rRNA in vivo as evaluated by reverse transcription PCR analysis on total F. septica RNA. Furthermore, RNA processing analyses in vitro and in vivo showed that 10 of 12 introns perform hydrolytic cleavage at the 3′ splice site, as well as intron circularization. Full-length intron RNA circles were detected in vivo. The order of splicing was analyzed by a reverse transcription PCR approach on cellular RNA, but no strict order of intron excision could be detected. Phylogenetic analysis indicated that most Fuligo introns were distantly related to each other and were independently gained in ribosomal DNA during evolution.

All group I intron sequences known in the nucleus interrupt the ribosomal RNA genes of protists and fungi, and more than 1,300 examples at 50 different integration sites are known ( Cannone et al. 2002 Jackson et al. 2002). Group I introns are characterized by conserved secondary RNA structures consisting of at least 10 paired segments (P1 to P10) common to most group I ribozymes, and usually several optional segments (P11 to P17) present in subsets of introns ( Lehnert et al. 1996 Einvik et al. 1998). The highly conserved catalytic core responsible for the self-splicing reaction consists of P4 to P6 and P3 to P9 ( Michel and Westhof 1990 Golden et al. 1998). Group I intron RNAs have been divided into 12 subclasses within five main groups (IA1-3, IB1-4, IC1-3, ID, and IE), based on conserved secondary structure, tertiary interactions, and phylogenetic analyses (see Michel and Westhof [1990] and Suh, Jones, and Blackwell [1999]). Group I intron RNAs are ribozymes catalyzing their own splicing reactions, resulting in perfectly ligated exons. The self-splicing reaction is initiated by a nucleophilic attack of an exogenous guanosine at the 5′ splice site and proceeds by a second transesterification reaction between the generated free 3′ end of the 5′ exon and the 3′ splice site ( Cech and Herschlag 1996). A parallel, competing, intron RNA–processing reaction, known as the intron circularization pathway, is initiated by hydrolytic cleavage at the 3′ splice site ( Nielsen et al. 2003). Here, the free 3′ hydroxyl group of the last intron residue (ωG) generated by hydrolysis attacks the 5′ exon-intron junction and results in the formation of full-length circular (FLC) intron RNAs, as well as nonligated exons. Thus, the circularization pathway generates nonfunctional rRNAs and appears to challenge the viability of the host.

It is generally considered that nuclear group I introns benefit from a ribosomal DNA localization because they are replicated as part of the host chromosomes and cotranscribed by RNA polymerase I as an integrated part of the pre-rRNA ( Lin and Vogt 1998 Jackson et al. 2002). Once transcribed, the group I introns have to be precisely spliced out to generate functional pre-rRNAs essential to host cell viability. The excised group I intron RNAs are either rapidly degraded ( Brehm and Cech 1983), reversed spliced into cognate or related RNA sites ( Roman and Woodson 1998), or, in the case of some mobile introns, further processed into homing endonuclease messenger RNAs ( Lin and Vogt 1998 Vader, Nielsen, and Johansen 1999 Decatur, Johansen, and Vogt 2000). The widespread distribution and patterns of relatedness suggest that group I introns are selfish genetic elements able to spread both vertically and horizontally between evolutionarily distinct lineages and are thought to be the product of multiple insertions and selective losses (see Goddard and Burt [1999]).

Despite the large number of nuclear group I introns known, very few have been subjected to experimentally tests for RNA processing activities in vitro as naked RNA and in vivo from endogenous pre-rRNAs. Among these are the Tetrahymena introns, their mobile cognate intron from Physarum, and the complex twin-ribozyme introns in Didymium and Naegleria. We recently reported that all these introns generate FLC intron RNAs when incubated in vitro ( Haugen, De Jonckheere, and Johansen 2002 Nielsen et al. 2003). Whereas the Tetrahymena introns do not generate detectable FLCs in vivo, the Didymium intron forms a significant amount of FLCs in the nucleus, thus challenging the viability of the cell. In this report we have characterized the small subunit (SSU) and large subunit (LSU) ribosomal DNA (rDNA) from Fuligo septica. The rDNA was found to contain 12 group I introns, which is the highest group I intron content reported for a single precursor rRNA species. Thus, F. septica serves as an attractive model system in the characterization of biological roles and molecular evolution of group I introns. Here, we present the results from molecular and phylogenetic analyses of the Fuligo introns to provide new insight into fundamental questions such as intron structure and function, intron-host biology, and intron origin and evolution.


Abstract

The exons of the self-splicing pre-ribosomal RNA of Tetrahymena thermophila are joined accurately in vitro, even when only 33 nucleotides of the natural 5′ exon and 38 nucleotides of the natural 3′ exon remain. RNA fingerprint analysis was used to identify the unique ribonuclease T1 oligonucleotide generated by exon ligation. Secondary digests of the ligation junction oligonucleotide with ribonuclease A confirmed the identity of the fragment and demonstrated that the phosphate group that forms the phosphodiester bond at the ligation junction is derived from the 5′ position of a uridine nucleotide in the RNA. This observation supports the prediction that the splice junction phosphate is derived from the 3′ splice site. These results emphasize the mechanistic similarities of RNA splicing reactions of the group I introns, group II introns and nuclear pre-mRNA introns.

This work was supported by NIH grant GM28039 to T. R. Cech.


Conclusions

Self-splicing, RNA structure and folding, and HE-dependent homing are fully described features of the group I introns in Tetrahymena and Physarum rDNA, but these studies represent only part of the story for nuclear group I introns. Additional studies have shown that there exist two main catalytic pathways for intron RNA: the intron splicing pathway and the intron FLC pathway. Intron homing is also represented by two distinct mechanisms: HE-dependent homing and the less efficient reverse-splicing-dependent homing. The latter mechanism sometimes results in intron insertion at non-allelic sites. The next important challenge is to understand the biological role of nuclear group I introns, and a first step has been achieved for the myxomycete protists, which appear to contain an abundance of diverse catalytic rDNA introns. Four main intron categories have been identified, from the true selfish HEG-containing and mobile group I introns, to introns that have become biochemically dependent on the host cell for splicing. Some introns appear obligatory for the host, and intron RNAs may evolve further to gain more regulatory functions. Finally, the lariat capping ribozyme (GIR1) is a unique example of a group I intron that has gained new catalytic properties and new biological roles in nuclear gene regulation.


Conclusions

The mechanisms that promote and prevent group I introns from proliferating among bacterial genomes are poorly understood, as is the long-term impact of introns on organismal viability. When present, it is assumed that introns are phenotypically neutral, yet the co-opting of intron functions by a riboswitch or the domestication of intron-encoded homing endonuclease as a regulatory protein (WhiA) indicates that introns can be a source of genetic novelty. Future research efforts directed at understanding the effect of group I introns on host gene expression, mechanisms of mobility to ectopic sites and their spread among bacterial genomes and phages will lead to valuable insights regarding the dynamics and evolution of group I introns.


The fate of an intervening sequence RNA: Excision and cyclization of the Tetrahymena ribosomal RNA intervening sequence in vivo

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The 3′ exon sequences are not essential for hydrolysis at the 3′-splice site

Sequences flanking both the Tetrahymena and the Physarum introns [ [17, 18] ] have been shown to influence on the rate of in vitro splicing. To test for similar effects of the 3′ exon on DiGIR2 hydrolytic cleavage at the 3′-SS, mutations were introduced into the eight first positions of the 3′-exon sequence (Fig. 2A) and analyzed in both the 5′-truncated and full-length splicing DiGIR2 contexts. Precursor (Pre) RNAs were incubated under splicing conditions in time course experiments and the generated RNA species were separated on 8 m urea/5% polyacrylamide gels. Compared to the wt exon context (Di347 Fig. 2A), no reductions in hydrolytic cleavage of truncated transcripts were observed even when 2–8 exon positions were changed (Di348–50). Di347 and Di350 precursor RNAs were subjected to more extensive time course experiments including quantification of radioactive decay from the gels using phosphoimager screens. Fraction hydrolyzed RNA (Cut) of the precursor was plotted vs. time (Fig. 2B) and fitted into a nonlinear first-order decay equation. The observed rate constants (kobs) are shown in Fig. 2B below the plot. Results indicate that the immediate 3′-exon sequence plays only a minor role in DiGIR2 hydrolysis, which corroborates the recent findings of the bacterial group IC3 ribozymes of Azoarcus and Synechococcus pre-tRNA [ [19] ]. Same mutational changes as in Di350 were introduced and tested in a DiGIR2 splicing context (DiGIR2.350). A time course experiment of DiGIR2.350 alongside the corresponding wild-type (DiGIR2.347) RNA is shown in Fig. 2C. The results indicate that the 3′ exon sequence is not important for DiGIR2 splicing (see RNA 5), but some reductions in hydrolytic cleavage at the 3′-SS (see RNA 3) and subsequent intron circle formation are observed (see RNA 1 and 6). This minor discrepancy between full-length splicing and 5′-truncated transcripts may be due to RNA interaction of the proposed P10 (Fig. 1B), which is present in the full-length splicing transcript but not the truncated transcript.

Analysis of DiGIR2 3′ exon sequences in hydrolytic cleavage and self-splicing. (A) Time course experiment (0–30 min) of 5′-truncated DiGIR2 containing different sequence substitution within the eight first positions of the 3′ exon. Mutant RNAs were subjected to splicing conditions [40 m m Tris pH 7.5, 10 m m MgCl, 200 m m KCl, 2 m m spermidine, 5 m m dithiothreitol, 0.2 m m GTP]. Exon nucleotides are presented as lower case letters and substituted positions are shaded. Pre, precursor RNA Cut, 5′ RNA product 3′SS, 3′-splice site. (B) Di347 and Di350 RNAs subjected to hydrolysis conditions (identical to splicing conditions but without GTP) and plotted as fraction uncleaved precursor (pre/total) vs. time. Curves were fitted to the nonlinear first-order decay equation F t = F4+F0 × ekobsxt and pseudo-first-order rate constants (kobs) were calculated. kobs variations represent differences between independent trials. RNA bands were quantitated by phosphoimager exposure with imagequant version 3.3 software. (C) Self-splicing time course experiments (0–30 min) of DiGIR2.350 and wild-type DiGIR2.347. DiGIR2.347 was constructed in order to generate a DiGIR2 equivalent to DiGIR2.350 (short-3′ exon sequences). Cir, intron RNA circle Pre, precursor RNA 5′-E, 5′ exon Int, Intron LE, ligated exons.


Abstract

NanGIR1 is a catalytic element inserted in the P6 loop of a group I intron (NanGIR2) in the small subunit rRNA precursor of the protist Naegleria andersoni [Einvik, C., Decatur, W. A., Embley, T. M., Vogt, V. M., and Johansen, S. (1997) RNA3, 710−720]. It catalyzes site-specific hydrolysis at an internal processing site (IPS) after a G residue that immediately follows the P9 stem−loop. Functional and structural analyses were initiated to compare NanGIR1 to group I introns that carry out self-splicing. Chemical modification and site-directed mutagenesis studies showed that NanGIR1 shares many structural elements with other group I introns, but also contains a pseudoknot (P15), which is important for catalytic activity. Deletion analysis revealed the boundaries of the minimum self-cleaving unit (178 nucleotides). The rate of self-cleavage was measured as a function of mono- and divalent ion concentration, temperature, and pH. The reaction at the IPS yields 5‘-phosphate and 3‘-hydroxyl termini, requires Mg 2+ or Mn 2+ ions, and is first-order in [OH - ] between pH 5.0 and 8.5. The latter results suggest that the nucleophile in the reaction is hydroxide or possibly a Mg 2+ -coordinated hydroxide. With a second-order rate constant of 1 × 10 5 min - 1 M - 1 , the self-cleavage reaction of NanGIR1 is 2 orders of magnitude faster than a similar site-specific hydrolysis reaction of the circular form of the Tetrahymena group I intron.

This work was supported in part by NIH Postdoctoral Grant GM18123 to E.J. and a Deutscher Akademischer Austauschdienst grant to S.A. S.A. is a Regensburg fellow. T.R.C. is an investigator of the Howard Hughes Medical Institute and an American Cancer Society Professor.

To whom correspondence should be addressed: Department of Chemistry and Biochemistry, Campus Box 215, University of Colorado, Boulder, CO 80309-0215. E-mail: [email protected]


7. Evolution of Improved trans -Splicing Group I Intron Ribozymes

7.3 mutations per ribozyme) led to a collapse of the evolving population, while 10 or 20 cycles of mutagenic PCR, (

2.4 and 4.8 mutations per ribozyme, respectively) led to stable populations. The effect of recombination [67] was tested with

1 recombination event per evolution round and ribozyme [66]. The most efficient, evolved motif did not benefit from recombination because it required four clustered mutations. However, recombination appeared to reduce deleterious mutations by at least four-fold. The evolved four mutations in the P6b loop of the ribozyme generated an accessible (C)5 homopentamer sequence (Figure 4B), which recruited the Rho transcription factor. Interestingly, the mutations did not increase trans -splicing efficiency or transcription efficiency in E. coli but strongly benefitted the translation of the spliced mRNA. This evolution illustrates how an evolving population of ribozymes can sample the interaction with cellular components and establish a novel binding site to those proteins that benefit its evolutionary interest.


THE FUTURE FOR RIBOZYMES

For ribozymes to become realistic therapeutic agents several obstacles need first to be overcome. These obstacles are the efficient delivery to a high percentage of the cell population, efficient expression of the ribozyme from a vector or intracellular ribozyme concentration, colocalization of the ribozyme with the target, specificity of ribozyme for the desired mRNA, and an enhancement of ribozyme-mediated substrate turnover.

Despite these reservations, results with ribozymes so far look promising, particularly in the HIV-1 studies. As our knowledge of RNA structure, secondary and tertiary, increases, we will be able to target the RNA more rationally, which may help with the problems of specificity. At the same time, the understanding of the physical localization of RNA in cells and its tracking as it moves from the nucleus to cytoplasm will also help in ensuring colocalization of the ribozyme and target. Modifications of the ribozymes, eg, the 2′ ribose with allyl group, increases the stability to nucleases quite dramatically. Similarly, DNA sequences allied to the ribozymes in use in our laboratory increase the stability. Entry into cells with liposomes or via vectors are also looking hopeful. Catalytic activity is maintained but research is still ongoing at tackling the problem of increasing the number of ribozyme molecules in proportion to the substrate molecules—a requirement for success. These molecules must retain their catalytic potential, must reach an accessible site in the substrate, and eventually be synthesized from the appropriate vector chosen for clinical trials. Work in the antisense DNA field would also benefit from solutions to these problems.

Supported by Grant No. 9667 of the Leukaemia Research Fund, London, UK.

Address reprint requests to Helen A. James, PhD, School of Biological Sciences, University of East Anglia, Norwich, Norfolk, NR4 7TJ UK.


Watch the video: Splicing (May 2022).